The choriocapillaris (CC) is a lobular, fenestrated vasculature that provides all of the oxygen and serum nutrients to the RPE and photoreceptors. We have elaborated the birth of human CC by hemo-vasculogenesis and the death of CC in wet and dry AMD in the last grant period. In dry AMD, CC cell death is related to loss of adjacent RPE. In wet AMD, loss of CC occurs in advance of choroidal neovascular membrane (CNV) formation, which is unexplained to date. We have observed high concentrations of serum proteins around CC in dry and wet AMD. The reason for this accumulation and influence of these proteins on CC is unknown. Although it is assumed that CC provides the nutrients for RPE and photoreceptors and removes waste, the exact mechanisms of transport are unknown. The proposed studies will characterize CC's normal transport mechanisms via fenestrations, caveolae, and coated pits. These mechanisms will be evaluated using gold nanoparticles of defined sizes, tagged serum proteins, and tagged serum lipids. We will determine if dysregulation in CC transport affects protein accumulation by using mice lacking caveolae (knock out cav-1, the major component in the caveolae system), or fenestrations (RPE-produced VEGF knocked out or neutralized), or mice with RPE over expressing VEGF, or producing basal laminar deposits. The proposed studies will determine the effects of Bruchs membrane deposits on CC transport and the effects of CC transport defects on deposit formation. We hypothesize that dysfunction in CC transport results in the serum protein accumulation in choroid we observe, which is toxic to CC and may be the reason that CC die in AMD. We will evaluate the toxicity of serum proteins (albumin, CRP, and alpha-2 macroglobulin with and without glycation) on choroidal endothelial cells (CEC) in vitro and determine if those serum proteins cause loss of tight junctions, changes in numbers of caveolae or fenestrations in CEC. It is also assumed that CC dysfunction is involved in AMD. Our preliminary studies demonstrated that loss in fenestrations is associated with Bruchs membrane deposits and RPE loss. In a large collection of AMD eyes that Greg Hageman has prepared for TEM, we will determine if CC fenestrations, caveolae, coated pits and/or tight junctions change in human AMD and the association of basal deposits, drusen, and RPE loss with these changes. In conclusion, this proposal will define the normal transport processes used by CC to supply nutrients to retina. We will also determine if these transport processes are altered in AMD. We will determine if serum proteins that accumulate with age in choroid contribute to dysfunction in CC transport and the death of CC we have observed in AMD. We will investigate how reduced or elevated VEGF changes CC transport. This new knowledge of CC transport and how it changes in AMD will be invaluable in developing new systemic therapeutic nanoparticles for preventing retinal degeneration and CNV that occurs in AMD. PUBLIC HEALTH RELEVANCE: This study will determine how the choriocapillaris (CC) transports nutrients to retinal pigment epithelium (RPE) and photoreceptors and if the CC transport systems are dysregulated in the presence of too little or too much VEGF. This will be accomplished in mice using uniform size and charge gold nanoparticles, and labeled albumin and lipoprotein to evaluate caveolae, fenestrations, coated pits, and tight junction. CC transport will also be evaluated in mice lacking fenestrations and caveolae, or with deposits on Bruch's membrane. These results will be compared to transmission electron microscopy of a well-characterized cohort of AMD and aged subjects. These studies will determine mechanisms of CC transport and how they are affected in AMD, both of which are unknown.